Engineering mouse cationic trypsinogen for rapid and selective activation by cathepsin B.

Area of Science:

• Biochemistry
• Molecular Biology
• Gastroenterology

Background:

• Intra-pancreatic trypsinogen activation is a key early event in pancreatitis.
• Trypsinogen activation occurs via autoactivation or by lysosomal cathepsin B (CTSB).
• Distinguishing these activation pathways is crucial for understanding pancreatitis.

Purpose of the Study:

• To biochemically characterize novel mouse cationic trypsinogen (isoform T7) mutants.
• To engineer a trypsinogen mutant selectively activated by CTSB but resistant to autoactivation.
• To establish a foundation for a preclinical model of CTSB-dependent pancreatitis.

Main Methods:

• Site-directed mutagenesis of mouse T7 trypsinogen.
• Biochemical assays to measure trypsinogen activation kinetics.
• Analysis of mutant sensitivity to CTSB and pH.

Main Results:

• Mutation K24G abolished autoactivation and enhanced CTSB activation 4-fold at pH 4.0.
• Mutation D22A accelerated CTSB-mediated activation by 2-fold.
• The combined D22A,K24G mutant showed 14-fold increased CTSB activation and normal pH sensitivity, with no autoactivation.

Conclusions:

• A novel mouse T7 trypsinogen mutant (D22A,K24G) was successfully engineered.
• This mutant is robustly activated by CTSB and resistant to autoactivation.
• These findings pave the way for a preclinical model of CTSB-dependent pancreatitis.